Lymphoma associated molecules and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated BBAP nucleic acid molecules, which encode proteins that interact with or bind to BAL molecules, which are differentially expressed in non-Hodgkin&#39;s lymphoma. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing BBAP nucleic acid molecules, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a BBAP gene has been introduced or disrupted. The invention still further provides isolated BBAP proteins, fusion proteins, antigenic peptides and anti-BBAP antibodies. Diagnostic methods using compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 09/957,635, filed on Sep. 19, 2001, which claimspriority to U.S. Provisional Application No. 60/233,791, filed on Sep.19, 2000, the entire contents of each application which are incorporatedherein by this reference.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, under grant 1P01CA66996-01A1 awarded by the NIH. The U.S. government therefore may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

The incidence of non-Hodgkin's lymphoma in the United States hasincreased by 75.1% between 1973 and 1992 (Kosary et al., SEER CancerStatistics Review, 1973-1992: Tables and Graphs, National CancerInstitute, NIH Publication No 96-2789, Bethesda, Md.: NIH 1995), apercentage increase exceeded only by that for prostate cancer, lungcancer in women, and melanoma.

Diffuse large B-cell lymphoma (DLB-CL) is the most common non-Hodgkin'slymphoma in adults. Although DLB-CL is curable in approximately 40% ofpatients, the majority of patients progress and die of their disease(Shipp et al. Non-Hodgkin's Lymphomas. In DeVita (ed): Principles andPractice of Oncology, 5th Edition, Philadelphia, J. B. LippincottCompany. pp. 2165-2220, 1997). Additional advancements in the treatmentof this aggressive but potentially curable non-Hodgkin's lymphoma arelikely to require a more precise understanding of the disease's cellularand molecular bases.

A novel gene termed “B-aggressive lymphoma” (BAL), was found to besignificantly more abundant in tumors from patients with “high-risk(HR)” (International Prognostic Index, IPI) fatal disease than in tumorsfrom cured “low risk (LR [IPI])” patients.

SUMMARY OF THE INVENTION

To identify other genes which contribute to the observed differences inclinical outcome in DLB-CLs, yeast two-hybrid screens (Zervos et al.(1993) Cell 72:223-232) were used to identify proteins which bind to orinteract with the BAL protein. A novel BAL-associated protein termed“B-lymphoma and BAL-associated protein” or “BBAP” has been identified,which specifically interacts with the BAL carboxyl terminal region. Theco-association between BBAP and BAL was confirmed by co-transfectingtagged constructs (FLAG-tagged BBAP and HA-tagged BAL) into COS cells,immunoprecipitating BBAP/BAL complexes with a FLAG antibody, blottingthe immunoprecipitates, and identifying BAL with an HA antibody.Moreover, paired northern blot analyses of BBAL and BAL transcripts havedemonstrated that these genes are expressed at very similar levels inmultiple normal tissues and a variety of hematopoietic cell lines.

Accordingly, in one aspect, the present invention provides isolatednucleic acid molecules encoding BBAP proteins or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of BBAP-encoding nucleicacids. The BBAP molecules of the present invention are useful asmodulating agents for regulating a variety of cellular processes.

In one embodiment, a BBAP nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or more identical to the nucleotide sequence (e.g., to the entire lengthof the nucleotide sequence) shown in SEQ ID NO:1 or 3.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof.In another embodiment, the nucleic acid molecule includes SEQ ID NO:3and nucleotides 1-2762 of SEQ ID NO:1. In another embodiment, thenucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-166 or2384-2762 of SEQ ID NO:1. In another preferred embodiment, the nucleicacid molecule consists of the nucleotide sequence shown in SEQ ID NO:1or 3.

In another preferred embodiment, the nucleic acid molecule includes afragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750 or more nucleotides (e.g., 50, 100, 200, 250 ormore contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:1or 3, or a complement thereof.

In another embodiment, a BBAP nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently homologous to the amino acid sequence of SEQ ID NO:2. In apreferred embodiment, a BBAP nucleic acid molecule includes a nucleotidesequence encoding a protein having an amino acid sequence at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or moreidentical to the entire length of the amino acid sequence of SEQ IDNO:2.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of human BBAP. In yet another preferredembodiment, the nucleic acid molecule includes a nucleotide sequenceencoding a protein having the amino acid sequence of SEQ ID NO:2. In yetanother preferred embodiment, the nucleic acid molecule is at least 300,400, 500, or more nucleotides in length. In a further preferredembodiment, the nucleic acid molecule is at least 600, 700, 800, 900, ormore nucleotides in length and encodes a protein having a BBAP activity(as described herein).

Another embodiment of the invention features nucleic acid molecules,preferably BBAP nucleic acid molecules, which specifically detect BBAPnucleic acid molecules relative to nucleic acid molecules encodingnon-BBAP proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 300, 350, 400, 500, 550, 600, or more nucleotidesin length and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence shown in SEQ ID NO:1 or 3 ora complement thereof.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or 3 understringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a BBAP nucleic acid molecule, e.g., thecoding strand of a BBAP nucleic acid molecule.

Another aspect of the invention provides a vector comprising a BBAPnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. In yetanother embodiment, the invention provides a host cell containing anucleic acid molecule of the invention. The invention also provides amethod for producing a protein, preferably a BBAP protein, by culturingin a suitable medium, a host cell, e.g., a mammalian host cell such as anon-human mammalian cell, of the invention containing a recombinantexpression vector, such that the protein is produced.

Another aspect of this invention features isolated or recombinant BBAPproteins and polypeptides. In one embodiment, the isolated protein,preferably a BBAP protein, includes at least one or more of thefollowing domains: a nuclear localization signal or a C3HC4-type zincfinger motif. In a preferred embodiment, a BBAP protein, includes atleast one or more of the following domains: a nuclear localizationsignal or a C3HC4-type zinc finger motif and has an amino acid sequenceat least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or more identical to the amino acid sequence of SEQ ID NO:2. Inanother preferred embodiment, a BBAP protein, includes at least one ormore of the following domains: a nuclear localization signal or aC3HC4-type zinc finger motif and plays a role in the pathogenesis ofnon-Hodgkin's lymphoma. In yet another preferred embodiment, a BBAPprotein, includes at least one or more of the following domains: anuclear localization signal or a C3HC4-type zinc finger motif and isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1 or 3.

In another embodiment, the invention features BBAP proteins which areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a BBAP protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques, based on the amino acidsequence of SEQ ID NO:2.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:2, wherein the fragmentcomprises at least 15 amino acids (e.g., contiguous amino acids) of theamino acid sequence of SEQ ID NO:2. In another embodiment, a BBAPprotein has the amino acid sequence of SEQ ID NO:2.

In another embodiment, the invention features an isolated BBAP proteinwhich is encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or more identical to a nucleotide sequence of SEQ IDNO:1 or 3. This invention further features an isolated BBAP proteinwhich is encoded by a nucleic acid molecule consisting of a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1or 3.

The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-BBAP polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind the BBAPproteins of the invention. In addition, the BBAP proteins orbiologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a BBAP nucleic acid molecule, protein or polypeptide ina biological sample by contacting the biological sample with an agentcapable of detecting a BBAP nucleic acid molecule, protein orpolypeptide such that the presence of a BBAP nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of BBAP activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofBBAP activity such that the presence of BBAP activity is detected in thebiological sample.

In another aspect, the invention provides a method for modulating BBAPactivity comprising contacting a cell capable of expressing BBAP with anagent that modulates BBAP activity such that BBAP activity in the cellis modulated. In one embodiment, the agent inhibits BBAP activity. Inanother embodiment, the agent stimulates BBAP activity. In oneembodiment, the agent is an antibody that specifically binds to a BBAPprotein. In another embodiment, the agent modulates expression of BBAPby modulating transcription of a BBAP gene or translation of a BBAPmRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand of aBBAP mRNA or a BBAP gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant BBAP proteinor nucleic acid expression or activity, e.g., non-Hodgkin's lymphoma, byadministering an agent which is a BBAP modulator to the subject. In oneembodiment, the BBAP modulator is a BBAP protein. In another embodimentthe BBAP modulator is a BBAP nucleic acid molecule. In yet anotherembodiment, the BBAP modulator is a peptide, peptidomimetic, or othersmall molecule. In a preferred embodiment, the disorder characterized byaberrant BBAP protein or nucleic acid expression is a proliferativedisorder, e.g., non-Hodgkin's lymphoma.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aBBAP protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a BBAP protein, wherein a wild-typeform of the gene encodes a protein with a BBAP activity.

In another aspect the invention provides a method for producing oridentifying a compound that binds to or modulates the activity of a BBAPprotein, by providing an indicator composition comprising a BBAP proteinhaving BBAP activity, contacting the indicator composition with a testcompound, and determining the effect of the test compound on BBAPactivity in the indicator composition to produce or identify a compoundthat modulates the activity of a BBAP protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence and predicted amino acid sequence ofhuman BBAP. The nucleotide sequence corresponds to nucleic acids 1 to2762 of SEQ ID NO:1. The amino acid sequence corresponds to amino acids1 to 739 of SEQ ID NO: 2. The coding region without the 5′ and 3′untranslated regions of the human BBAP gene is shown in SEQ ID NO:3.

FIG. 2 depicts the results from a paired northern blot analysis of BBAPand BAL transcripts, which indicate that these genes are expressed atvery similar levels in multiple normal tissues.

FIG. 3 depicts a schematic diagram of the BAL fragments that were usedas baits in the yeast two-hybrid assay.

FIG. 4 depicts the results from an expression assay which demonstratesthat the BBAP protein has an inhibitory effect on E47 activity. 293cells were co-transfected with [E5+E2]₆TATA-Luc (50 ng) andnull-Renillla luciferase control reporter construct (5 ng), theindicated amounts (Ong, 25 ng, or 50 ng) of the E47mig plasmid encodingE47, and the indicated amounts (Ong, 25 ng, or 50 ng) of the pcDNA-Mycplasmid encoding DTX1 or/and pFLAG-CMV plasmid encoding BBAP. Cellextracts were prepared 44 hours post-transfection. E2+E5 reporterluciferase activity, corrected for null-Renillar luciferase activity, isexpressed as fold activation relative to control 293 cells notexpressing E47, DTX1 and BBAP. Error bars indicate standard deviation oftwo independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as BBAP nucleic acid and proteinmolecules. The BBAP proteins of the present invention bind to orinteract with the BAL molecules which are differentially expressed inmalignancies such as lymphoma, e.g., non-Hodgkin's lymphoma. The newlyidentified BBAP nucleic acid and protein molecules can be used toidentify cells exhibiting or predisposed to a malignancy such aslymphoma, e.g., non-Hodgkin's lymphoma, thereby diagnosing subjectshaving, or prone to developing such disorders.

As used herein, a “malignancy” includes a cancerous uncontrolled growthof cells in an area of the body. Malignant cancers are typicallyclassified by their microscopic appearance and the type of tissue fromwhich they arise. Examples of malignancies include carcinomas, sarcomas,myelomas, chondrosarcomas, adenosarcomas, angiosarcomas, neuroblastomas,gliomas, medulloblastomas, erythroleukemias, and myelogenous leukemias.

As used herein, a “lymphoma” includes a malignant neoplastic disorder oflymphoreticular tissue which produces a distinct tumor mass. Lymphomasinclude tumors derived from the lymphoid lineage. Lymphomas usuallyarise in lymph nodes, the spleen, or other areas rich in lymphoidtissue. Lymphomas are typically subclassified as Hodgkin's disease andNon-Hodgkin's lymphomas, e.g., Burkitt's lymphoma, large-cell lymphoma,and follicular lymphoma.

As used herein, “differential expression” or differentially expressed”includes both quantitative as well as qualitative differences in thetemporal and/or cellular expression pattern of a gene, e.g., the BBAPgene, among, for example, normal cells and cells from patients with“high risk” fatal diffuse large B-cell lymphoma (DLB-CL) or “low risk”cured DLB-CL. Genes which are differentially expressed can be used aspart of a prognostic or diagnostic marker for the evaluation of subjectsat risk for developing a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma. Depending on the expression level of the gene,the progression state or the aggressiveness of the disorder can beevaluated. Methods for detecting the differential expression of a geneare described herein.

The BBAP molecules comprise a family of molecules having certainconserved structural and functional features. The term “family” whenreferring to the protein and nucleic acid molecules of the invention isintended to mean two or more proteins or nucleic acid molecules having acommon structural domain or motif and having sufficient amino acid ornucleotide sequence homology as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof human origin, as well as other, distinct proteins of human origin oralternatively, can contain homologues of non-human origin. Members of afamily may also have common functional characteristics.

For example, the family of BBAP proteins comprise at least one “nuclearlocalization signal.” As used herein, the term “nuclear localizationsignal” includes an amino acid sequence of about 4-20 amino acidresidues in length, which serves to direct a protein to the nucleus.Typically, the nuclear localization sequence is rich in basic aminoacids. A nuclear localization signal may have one or more of thefollowing sequences: RKRH (SEQ ID NO:5); PRVRRKL (SEQ ID NO:6); orRKHLHQTKFADDFRKRH (SEQ ID NO:7). Nuclear localization signals aredescribed in, for example, Gorlich D. (1998) EMBO 5.17:2721-7, thecontents of which are incorporated herein by reference. Amino acidresidues 20-26, 462-478, and 475-478 of the human BBAP comprise nuclearlocalization signals.

In another embodiment, a BBAP protein of the present invention isidentified based on the presence of at least one “C3HC4-type zinc fingermotif” in the protein or corresponding nucleic acid molecule. As usedherein, the term “C3HC4-type zinc finger motif” includes an amino acidsequence of about 40-70 amino acid residues in length and having thegeneral sequenceC-X-(I,V)-C-X(11-30)-C-X-H-X-(F,I,L)-C-X(2)-C-(I,L,M)-X(10-18)-C-P-X-C(SEQ ID NO:4) (where X can be any amino acid). Proteins comprising aring-H₂-finger motif are believed to interact with DNA and to beinvolved in diverse functions, including site specific recombination,DNA repair, and transcriptional regulation. The ring-H₂-finger may alsobind zinc/divalent metal ions to form a structure that is involved inspecific protein-protein interactions (similar to the zinc-cysteineclusters of the adenovirus E1A). Amino acid residues 561-599 of thehuman BBAP comprise a C3HC4-type zinc finger motif.

Isolated BBAP proteins of the invention have an amino acid sequencesufficiently homologous to the amino acid sequence of SEQ ID NO:2 or areencoded by a nucleotide sequence sufficiently homologous to SEQ ID NO:1or 3. As used herein, the term “sufficiently homologous” refers to afirst amino acid or nucleotide sequence which contains a sufficient orminimum number of identical or equivalent (e.g., an amino acid residuewhich has a similar side chain) amino acid residues or nucleotides to asecond amino acid or nucleotide sequence such that the first and secondamino acid or nucleotide sequences share common structural domains ormotifs and/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains have at least30%, 40%, or 50%, preferably 60% identity, more preferably 70%-80%, andeven more DFN-036 preferably 90-95% identity across the amino acidsequences of the domains are defined herein as sufficiently identical.Furthermore, amino acid or nucleotide sequences which share at least30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95%identity and share a common functional activity are defined herein assufficiently identical.

As used interchangeably herein, “BBAP activity”, “biological activity ofBBAP” or “functional activity of BBAP”, refers to an activity exerted bya BBAP protein, polypeptide or nucleic acid molecule on a BBAPresponsive cell or on a BBAP protein substrate, as determined in vivo,ex vivo, or in vitro, according to standard techniques. In oneembodiment, a BBAP activity is a direct activity, such as an associationwith a BBAP-target molecule, e.g., a BAL molecule. As used herein, a“target molecule” or “binding partner” is a molecule with which a BBAPprotein binds or interacts in nature, such that BBAP-mediated functionis achieved. A BBAP target molecule can be a non-BBAP molecule such as aBAL molecule. In an exemplary embodiment, a BBAP target molecule is aBBAP ligand. Alternatively, a BBAP activity is an indirect activity,such as a cellular signaling activity mediated by interaction of theBBAP protein with a BBAP ligand. BBAP activities include modulation ofcellular adhesion and modulation of the aggressiveness or severity of amalignancy such as DLB-CL. BBAP activities are described herein.

Accordingly, another embodiment of the invention features isolated BBAPproteins and polypeptides having a BBAP activity. Preferred proteins areBBAP proteins having at least one or more of the following domains: anuclear localization signal or a C3HC4-type zinc finger motif and,preferably, a BBAP activity.

The nucleotide sequence of the isolated human BBAP cDNA and thepredicted amino acid sequence of the human BBAP polypeptide are shown inFIG. 1 and in SEQ ID NOS:1 and 2, respectively.

The human BBAP gene, which is approximately 2762 nucleotides in length,encodes a protein having a molecular weight of approximately 83.6 kD andwhich is approximately 739 amino acid residues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode BBAP proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify BBAP-encoding nucleic acid molecules (e.g., BBAPmRNA) and fragments for use as PCR primers for the amplification ormutation of BBAP nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated BBAP nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1 or 3 or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or portion of thenucleic acid sequence of SEQ ID NO:1 or 3, as a hybridization probe,BBAP nucleic acid molecules can be isolated using standard hybridizationand cloning techniques (e.g., as described in Sambrook, J., Fritsh, E.F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1 or 3 can be isolated by the polymerase chain reaction (PCR)using synthetic oligonucleotide primers designed based upon the sequenceof SEQ ID NO:1 or 3.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to BBAP nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1. Thesequence of SEQ ID NO:1 corresponds to the human BBAP cDNA. This cDNAcomprises sequences encoding the human BBAP protein (i.e., “the codingregion”, from nucleotides 167-2383), as well as 5′ untranslatedsequences (nucleotides 1-166) and 3′ untranslated sequences (nucleotides2384-2762). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:1 (e.g., nucleotides 167-2383,corresponding to SEQ ID NO:3).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1 or 3, or a portion of anyof these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1 or 3, isone which is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1 or 3, such that it can hybridize to the nucleotidesequence shown in SEQ ID NO:1 or 3.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or more identical to the entire length of the nucleotide sequenceshown in SEQ ID NO:1 or 3 or a portion of any of these nucleotidesequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1 or 3, for example, afragment which can be used as a probe or primer or a fragment encoding aportion of a BBAP protein, e.g., a biologically active portion of a BBAPprotein. The nucleotide sequence determined from the cloning of the BBAPgene allows for the generation of probes and primers designed for use inidentifying and/or cloning other BBAP family members, as well as BBAPhomologues from other species. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO:1 or 3, of ananti-sense sequence of SEQ ID NO:1 or 3, or of a naturally occurringallelic variant or mutant of SEQ ID NO:1 or 3. In an exemplaryembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which is greater than 300-350, 350-400, 400-450,452, 452-500, 500-550, 550-600, 607, 607-650, 650-700, 700-750, 750-800,800-850, 850-900, 900-950, 950-1000, or more nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:1 or 3. Ranges intermediate to the above-recitedvalues are also intended to be part of this invention. For example,ranges using any combination of the above recited values as upper and/orlower limits are intended to be included.

Probes based on the BBAP nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a BBAP protein, such as by measuring a level ofa BBAP-encoding nucleic acid in a sample of cells from a subject e.g.,detecting BBAP mRNA levels or determining whether a genomic BBAP genehas been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aBBAP protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1 or 3, which encodes a polypeptide having a BBAPbiological activity (the biological activities of the BBAP proteins aredescribed herein), expressing the encoded portion of the BBAP protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the BBAP protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1 or 3, due todegeneracy of the genetic code and thus encode the same BBAP proteins asthose encoded by the nucleotide sequence shown in SEQ ID NO:1 or 3. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a protein having an amino acidsequence shown in SEQ ID NO:2.

In addition to the BBAP nucleotide sequences shown in SEQ ID NO:1 or 3,it will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theBBAP proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the BBAP genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a BBAPprotein, preferably a mammalian BBAP protein, and can further includenon-coding regulatory sequences, and introns.

Allelic variants of human BBAP include both functional andnon-functional BBAP proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the human BBAP protein thatmaintain the ability to bind a BBAP ligand and/or modulate theoccurrence or severity of a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma. Functional allelic variants will typicallycontain only conservative substitution of one or more amino acids of SEQID NO:2 or substitution, deletion or insertion of non-critical residuesin non-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human BBAP protein that do not have the abilityto either bind a BBAP ligand and/or modulate occurrence or severity of amalignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma.Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:2 or a substitution,insertion or deletion in critical residues or critical regions.

The present invention further provides non-human orthologues of thehuman BBAP protein. Orthologues of the human BBAP protein are proteinsthat are isolated from non-human organisms and possess the same BBAPligand binding and/or modulation of the occurrence or severity of amalignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma capabilitiesof the human BBAP protein. Orthologues of the human BBAP protein canreadily be identified as comprising an amino acid sequence that issubstantially homologous to SEQ ID NO:2.

Moreover, nucleic acid molecules encoding other BBAP family members and,thus, which have a nucleotide sequence which differs from the BBAPsequences of SEQ ID NO:1 or 3 are intended to be within the scope of theinvention. For example, another BBAP cDNA can be identified based on thenucleotide sequence of human BBAP. Moreover, nucleic acid moleculesencoding BBAP proteins from different species, and which, thus, have anucleotide sequence which differs from the BBAP sequences of SEQ ID NO:1or 3 are intended to be within the scope of the invention. For example,a monkey or murine BBAP cDNA can be identified based on the nucleotidesequence of a human BBAP.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the BBAP cDNAs of the invention can be isolated based ontheir homology to the BBAP nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the BBAP cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the BBAP gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1 or 3. In otherembodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 250,300, 350, 400, 450, 452, 500, 550, 607, 600, 650, 700, 750, 800, 850,900, or 950 nucleotides in length. As used herein, the term “hybridizesunder stringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%identical to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 70%,more preferably at least about 80%, even more preferably at least about85% or 90% identical to each other typically remain hybridized to eachother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., morepreferably at 60° C., and even more preferably at 65° C. Preferably, anisolated nucleic acid molecule of the invention that hybridizes understringent conditions to the sequence of SEQ ID NO:1 or 3 corresponds toa naturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

In addition to naturally-occurring allelic variants of the BBAPsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1 or 3 thereby leading to changes inthe amino acid sequence of the encoded BBAP proteins, without alteringthe functional ability of the BBAP proteins. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:1 or 3. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of BBAP (e.g., the sequence of SEQ ID NO:2)without altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are conserved among the BBAP proteins of the presentinvention, are predicted to be particularly unamenable to alteration.Furthermore, additional amino acid residues that are conserved betweenthe BBAP proteins of the present invention and other members of the BBAPfamily are not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding BBAP proteins that contain changes in amino acidresidues that are not essential for activity. Such BBAP proteins differin amino acid sequence from SEQ ID NO:2, yet retain biological activity.In one embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or more homologous to SEQ ID NO:2.

An isolated nucleic acid molecule encoding a BBAP protein homologous tothe protein of SEQ ID NO:2 can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO:1 or 3, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO:1 or 3, by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a BBAP protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a BBAP coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened forBBAP biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1 or 3, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

In a preferred embodiment, a mutant BBAP protein can be assayed for theability to (1) interact with a non-BBAP protein molecule, e.g., BAL, (2)activate a BBAP-dependent signal transduction pathway, (3) modulate theoccurrence or severity of a lymphoma, e.g., non Hodgkin's lymphoma, (4)bind zinc, (5) bind DNA, (6) localize to the nucleus, or (7) modulatethe migration of malignant cells, e.g., B-lymphoma cells.

In addition to the nucleic acid molecules encoding BBAP proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire BBAP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding BBAP. Theterm “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of human and murine BBAP corresponds to SEQ ID NO:3).In another embodiment, the antisense nucleic acid molecule is antisenseto a “noncoding region” of the coding strand of a nucleotide sequenceencoding BBAP. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding BBAP disclosed herein (e.g.,SEQ ID NO:3), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof BBAP mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of BBAPmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of BBAP mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galaciosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a BBAP proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveBBAP mRNA transcripts to thereby inhibit translation of BBAP mRNA. Aribozyme having specificity for a BBAP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a BBAP cDNA disclosedherein (i.e., SEQ ID NO:1 or 3). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a BBAP-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,BBAP mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, BBAP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the BBAP(e.g., the BBAP promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the BBAP gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

In yet another embodiment, the BBAP nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & MedicinalChemistry 4 (1): 5-23). As used herein, the terms “peptide nucleicacids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, inwhich the deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe etal. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of BBAP nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of BBAP nucleic acid molecules can also be used in theanalysis of single base pair mutations in a gene, (e.g., by PNA-directedPCR clamping); as ‘artificial restriction enzymes’ when used incombination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996)supra)); or as probes or primers for DNA sequencing or hybridization(Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of BBAP can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of BBAP nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. (1996) supra and Finn P. J. et al.(1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain canbe synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

II. Isolated BBAP Proteins and Anti-BBAP Antibodies

One aspect of the invention pertains to isolated BBAP proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-BBAP antibodies. In oneembodiment, native BBAP proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BBAP proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a BBAP protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theBBAP protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of BBAPprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of BBAP protein having less than about 30% (by dryweight) of non-BBAP protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-BBAP protein,still more preferably less than about 10% of non-BBAP protein, and mostpreferably less than about 5% non-BBAP protein. When the BBAP protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BBAP protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of BBAP protein having less than about 30% (by dry weight)of chemical precursors or non-BBAP chemicals, more preferably less thanabout 20% chemical precursors or non-BBAP chemicals, still morepreferably less than about 10% chemical precursors or non-BBAPchemicals, and most preferably less than about 5% chemical precursors ornon-BBAP chemicals.

As used herein, a “biologically active portion” of a BBAP proteinincludes a fragment of a BBAP protein which participates in aninteraction between a BBAP molecule and a non-BBAP molecule.Biologically active portions of a BBAP protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the BBAP protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include less amino acids than thefull length BBAP proteins, and exhibit at least one activity of a BBAPprotein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the BBAP protein, e.g., modulatingcellular adhesion. A biologically active portion of a BBAP protein canbe a polypeptide which is, for example, 10, 25, 50, 100, 200 or moreamino acids in length. Biologically active portions of a BBAP proteincan be used as targets for developing agents which modulate a BBAPmediated activity, e.g., the occurrence or severity of a lymphoma, e.g.,non-Hodgkin's lymphoma.

In one embodiment, a biologically active portion of a BBAP proteincomprises at least one nuclear localization signal and/or at least oneC3HC4-type zinc finger motif. It is to be understood that a preferredbiologically active portion of a BBAP protein of the present inventionmay contain at least one of the above-identified structural domains. Amore preferred biologically active portion of a BBAP protein may containat least two of the above-identified structural domains. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native BBAP protein.

In a preferred embodiment, the BBAP protein has an amino acid sequenceshown in SEQ ID NO:2. In other embodiments, the BBAP protein issubstantially homologous to SEQ ID NO:2, and retains the functionalactivity of the protein of SEQ ID NO:2, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. Accordingly, in another embodiment, theBBAP protein is a protein which comprises an amino acid sequence atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or more identical to SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the BBAP amino acidsequence of SEQ ID NO:2 having 177 amino acid residues, at least 80,preferably at least 100, more preferably at least 120, even morepreferably at least 140, and even more preferably at least 150, 160 or170 amino acid residues are aligned). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available at theGenetics Computer Group website), using either a Blosum 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat the Genetics Computer Group website), using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6. In another embodiment, the percent identity between twoamino acid or nucleotide sequences is determined using the algorithm ofE. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0 or 2.0U), using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to BBAP nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to BBAP proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the NCBI website.

The invention also provides BBAP chimeric or fusion proteins. As usedherein, a BBAP “chimeric protein” or “fusion protein” comprises a BBAPpolypeptide operatively linked to a non-BBAP polypeptide. An “BBAPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to BBAP, whereas a “non-BBAP polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the BBAP protein, e.g., aprotein which is different from the BBAP protein and which is derivedfrom the same or a different organism. Within a BBAP fusion protein theBBAP polypeptide can correspond to all or a portion of a BBAP protein.In a preferred embodiment, a BBAP fusion protein comprises at least onebiologically active portion of a BBAP protein. In another preferredembodiment, a BBAP fusion protein comprises at least two biologicallyactive portions of a BBAP protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the BBAP polypeptideand the non-BBAP polypeptide are fused in-frame to each other. Thenon-BBAP polypeptide can be fused to the N-terminus or C-terminus of theBBAP polypeptide.

For example, in one embodiment, the fusion protein is a GST-BBAP fusionprotein in which the BBAP sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant BBAP.

In another embodiment, the fusion protein is a BBAP protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of BBAP can beincreased through use of a heterologous signal sequence.

The BBAP fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. TheBBAP fusion proteins can be used to affect the bioavailability of a BBAPsubstrate. Use of BBAP fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a BBAP protein; (ii)mis-regulation of the BBAP gene; and (iii) aberrant post-translationalmodification of a BBAP protein.

Moreover, the BBAP-fusion proteins of the invention can be used asimmunogens to produce anti-BBAP antibodies in a subject, to purify BBAPligands and in screening assays to identify molecules which inhibit theinteraction of BBAP with a BBAP substrate.

Preferably, a BBAP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnBBAP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the BBAP protein.

The present invention also pertains to variants of the BBAP proteinswhich function as either BBAP agonists (mimetics) or as BBAPantagonists. Variants of the BBAP proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a BBAPprotein. An agonist of the BBAP proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a BBAP protein. An antagonist of a BBAP protein caninhibit one or more of the activities of the naturally occurring form ofthe BBAP protein by, for example, competitively modulating aBBAP-mediated activity of a BBAP protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the BBAP protein.

In one embodiment, variants of a BBAP protein which function as eitherBBAP agonists (mimetics) or as BBAP antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a BBAP protein for BBAP protein agonist or antagonist activity. Inone embodiment, a variegated library of BBAP variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of BBAP variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential BBAP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of BBAP sequences therein. There are avariety of methods which can be used to produce libraries of potentialBBAP variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential BBAP sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of a BBAP protein coding sequencecan be used to generate a variegated population of BBAP fragments forscreening and subsequent selection of variants of a BBAP protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a BBAP coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the BBAP protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of BBAP proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify BBAP variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated BBAP library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to aparticular ligand in a BBAP-dependent manner. The transfected cells arethen contacted with the ligand and the effect of expression of themutant on signaling by the ligand can be detected, e.g., by measuringcell survival or the activity of a BBAP-regulated transcription factor.Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of signaling by the ligand,and the individual clones further characterized.

An isolated BBAP protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind BBAP using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length BBAP protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of BBAP for use as immunogens. Theantigenic peptide of BBAP comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2 and encompasses an epitopeof BBAP such that an antibody raised against the peptide forms aspecific immune complex with BBAP. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofBBAP that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity.

A BBAP immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed BBAP protein or a chemicallysynthesized BBAP polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic BBAP preparation induces a polyclonal anti-BBAP antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-BBAPantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as BBAP.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind BBAP. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of BBAP. A monoclonal antibody composition thustypically displays a single binding affinity for a particular BBAPprotein with which it immunoreacts.

Polyclonal anti-BBAP antibodies can be prepared as described above byimmunizing a suitable subject with a BBAP immunogen. The anti-BBAPantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized BBAP. If desired, the antibody moleculesdirected against BBAP can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-BBAP antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 0.255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a BBAP immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds BBAP.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-BBAP monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindBBAP, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-BBAP antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with BBAP to thereby isolateimmunoglobulin library members that bind BBAP. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J.Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gramet al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. AcidRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-BBAP antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-BBAP antibody (e.g., monoclonal antibody) can be used to isolateBBAP by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-BBAP antibody can facilitate thepurification of natural BBAP from cells and of recombinantly producedBBAP expressed in host cells. Moreover, an anti-BBAP antibody can beused to detect BBAP protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the BBAP protein. Anti-BBAP antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, -galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a BBAP protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology. Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., BBAP proteins,mutant forms of BBAP proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of BBAP proteins in prokaryotic or eukaryotic cells. Forexample, BBAP proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in BBAP activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for BBAP proteins, for example. In apreferred embodiment, a BBAP fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn 10-lac fusion promoter mediated bya coexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the BBAP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, BBAP proteins can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to BBAP mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which a BBAPnucleic acid molecule of the invention is introduced, e.g., a BBAPnucleic acid molecule within a recombinant expression vector or a BBAPnucleic acid molecule containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aBBAP protein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a BBAP protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a BBAP protein.Accordingly, the invention further provides methods for producing a BBAPprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a BBAP protein has beenintroduced) in a suitable medium such that a BBAP protein is produced.In another embodiment, the method further comprises isolating a BBAPprotein from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichBBAP-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous BBAPsequences have been introduced into their genome or homologousrecombinant animals in which endogenous BBAP sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a BBAP and for identifying and/or evaluating modulators ofBBAP activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous BBAP gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing aBBAP-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The BBAPcDNA sequence of SEQ ID NO:1 can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of ahuman BBAP gene, such as a mouse or rat BBAP gene, can be used as atransgene. Alternatively, a BBAP gene homologue, can be isolated basedon hybridization to the BBAP cDNA sequences of SEQ ID NO:1 or 3(described further in subsection I above) and used as a transgene.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to aBBAP transgene to direct expression of a BBAP protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a BBAP transgene in its genome and/or expression of BBAPmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a BBAPprotein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a BBAP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the BBAP gene. The BBAP gene can be a human gene(e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non-humanhomologue of a human BBAP gene. For example, a mouse BBAP gene can beused to construct a homologous recombination nucleic acid molecule,e.g., a vector, suitable for altering an endogenous BBAP gene in themouse genome. In a preferred embodiment, the homologous recombinationnucleic acid molecule is designed such that, upon homologousrecombination, the endogenous BBAP gene is functionally disrupted (i.e.,no longer encodes a functional protein; also referred to as a “knockout” vector). Alternatively, the homologous recombination nucleic acidmolecule can be designed such that, upon homologous recombination, theendogenous BBAP gene is mutated or otherwise altered but still encodesfunctional protein (e.g., the upstream regulatory region can be alteredto thereby alter the expression of the endogenous BBAP protein). In thehomologous recombination nucleic acid molecule, the altered portion ofthe BBAP gene is flanked at its 5′ and 3′ ends by additional nucleicacid sequence of the BBAP gene to allow for homologous recombination tooccur between the exogenous BBAP gene carried by the homologousrecombination nucleic acid molecule and an endogenous BBAP gene in acell, e.g., an embryonic stem cell. The additional flanking BBAP nucleicacid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The homologous recombination nucleic acidmolecule is introduced into a cell, e.g., an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced BBAP genehas homologously recombined with the endogenous BBAP gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells canthen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage PI. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The recontructed oocyte is then cultured such that it develops to morulaor blastocyte and then transferred to pseudopregnant female fosteranimal. The offspring borne of this female foster animal will be a cloneof the animal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The BBAP nucleic acid molecules, fragments of BBAP proteins, andanti-BBAP antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a fragment of a BBAP protein or an anti-BBAP antibody)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a BBAP protein of the invention has one or more of thefollowing activities: (1) it interacts with a non-BBAP protein molecule,e.g., BAL; (2) it activates a BBAP-dependent signal transductionpathway; (3) it modulates the occurrence and severity of a malignancysuch as a lymphoma, e.g., non-Hodgkin's lymphoma; (4) it modulates cellmigration, motility, and shape, as well as cell/cell andcell/extra-cellular matrix interactions; (5) it modulates the activityof E47, and (6) it modulates the degradation of non-BBAP proteins (e.g.,BAL) by, for example, modifying these proteins and targeting them to theproteoliposome and, thus, can be used to, for example, (1) modulate theinteraction with a non-BBAP protein molecule, e.g., BAL; (2) activate aBBAP-dependent signal transduction pathway; (3) modulate the occurrenceand severity of a malignancy such as a lymphoma, e.g., non-Hodgkin'slymphoma; (4) modulate cell migration, motility, and shape, as well ascell/cell and cell/extra-cellular matrix interactions; (5) modulate theactivity of E47, and/or (6) modulate the degradation of non-BBAPproteins (e.g., BAL) by, for example, modifying these proteins andtargeting them to the proteoliposome.

The isolated nucleic acid molecules of the invention can be used, forexample, to express BBAP protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect BBAP mRNA(e.g., in a biological sample) or a genetic alteration in a BBAP gene,and to modulate BBAP activity, as described further below. The BBAPproteins can be used to treat disorders characterized by insufficient orexcessive production of a BBAP substrate or production of BBAPinhibitors. In addition, the BBAP proteins can be used to screen fornaturally occurring BBAP substrates, to screen for drugs or compoundswhich modulate BBAP activity, as well as to treat disorderscharacterized by insufficient or excessive production of BBAP protein orproduction of BBAP protein forms which have decreased or aberrantactivity compared to BBAP wild type protein (e.g., Non-Hodgkin'slymphoma). Moreover, the anti-BBAP antibodies of the invention can beused to detect and isolate BBAP proteins, regulate the bioavailabilityof BBAP proteins, and modulate BBAP activity.

A. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying and/or producing modulators, i.e., candidate ortest compounds or agents (e.g., peptides, peptidomimetics, smallmolecules or other drugs) which bind to BBAP proteins, have astimulatory or inhibitory effect on, for example, BBAP expression orBBAP activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a BBAP substrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a BBAP protein or polypeptideor biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a BBAP protein or polypeptideor biologically active portion thereof. The test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a BBAP protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate BBAP activity is determined. Determining the ability of thetest compound to modulate BBAP activity can be accomplished bymonitoring, for example, the survival of a cell which expresses BBAP orthe activity of a BBAP-regulated transcription factor. The cell, forexample, can be of mammalian origin, e.g., a peripheral blood cell.

The ability of the test compound to modulate BBAP binding to a substrateor to bind to BBAP can also be determined. Determining the ability ofthe test compound to modulate BBAP binding to a substrate can beaccomplished, for example, by coupling the BBAP substrate with aradioisotope or enzymatic label such that binding of the BBAP substrateto BBAP can be determined by detecting the labeled BBAP substrate in acomplex. Determining the ability of the test compound to bind BBAP canbe accomplished, for example, by coupling the compound with aradioisotope or enzymatic label such that binding of the compound toBBAP can be determined by detecting the labeled BBAP compound in acomplex. For example, compounds (e.g., BBAP substrates) can be labeledwith ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a BBAP substrate) to interact with BBAP without thelabeling of any of the interactants. For example, a microphysiometer canbe used to detect the interaction of a compound with BBAP without thelabeling of either the compound or the BBAP. McConnell, H. M. et al.(1992) Science 257:1906-1912. As used herein, a “microphysiometer”(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and BBAP.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a BBAP target molecule (e.g., a BBAPsubstrate such as BAL) with a test compound and determining the abilityof the test compound to modulate (e.g. stimulate or inhibit) theactivity of the BBAP target molecule. Determining the ability of thetest compound to modulate the activity of a BBAP target molecule can beaccomplished, for example, by determining the ability of the BBAPprotein to bind to or interact with the BBAP target molecule, e.g., BALor DNA.

Determining the ability of the BBAP protein or a biologically activefragment thereof, to bind to or interact with a BBAP target molecule canbe accomplished by one of the methods described above for determiningdirect binding. In a preferred embodiment, determining the ability ofthe BBAP protein to bind to or interact with a BBAP target molecule canbe accomplished by determining the activity of the target molecule. Forexample, the activity of the target molecule can be determined bydetecting induction of a cellular second messenger of the target (i.e.,intracellular Ca²⁺, diacylglycerol, IP₃, and the like), detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which a BBAP protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the BBAP protein or biologically active portionthereof is determined. Preferred biologically active portions of theBBAP proteins to be used in assays of the present invention includefragments which participate in interactions with non-BBAP molecules,e.g., fragments with high surface probability scores. Binding of thetest compound to the BBAP protein can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the BBAP protein or biologically active portionthereof with a known compound which binds BBAP to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a BBAP protein, whereindetermining the ability of the test compound to interact with a BBAPprotein comprises determining the ability of the test compound topreferentially bind to BBAP or biologically active portion thereof ascompared to the known compound.

In another embodiment, the assay is a cell-free assay in which a BBAPprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the BBAP protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a BBAP protein can beaccomplished, for example, by determining the ability of the BBAPprotein to bind to a BBAP target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the BBAP protein to bind to a BBAP target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a BBAP protein can be accomplishedby determining the ability of the BBAP protein to further modulate theactivity of a downstream effector of a BBAP target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins (e.g.,BBAP proteins or biologically active portions thereof). In the case ofcell-free assays in which a membrane-bound form of an isolated proteinis used it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of the isolated protein is maintained insolution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either BBAP or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a BBAP protein, or interaction of aBBAP protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/BBAP fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or BBAP protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of BBAPbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a BBAPprotein or a BBAP target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated BBAP protein ortarget molecules can be prepared from biotin-NHS(N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with BBAP protein or target molecules but which donot interfere with binding of the BBAP protein to its target moleculecan be derivatized to the wells of the plate, and unbound target or BBAPprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the BBAP protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the BBAP protein or target molecule.

In another embodiment, modulators of BBAP expression are produced oridentified in a method wherein a cell is contacted with a candidatecompound and the expression of BBAP mRNA or protein in the cell isdetermined. The level of expression of BBAP mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of BBAP mRNA or protein in the absence of the candidatecompound. The candidate compound can then be produced or identified as amodulator of BBAP expression based on this comparison. For example, whenexpression of BBAP mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofBBAP mRNA or protein expression (i.e. a stimulator of BBAP mRNA orprotein expression is produced). Alternatively, when expression of BBAPmRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is produced or identified as an inhibitor of BBAP mRNA orprotein expression (i.e. an inhibitor of BBAP mRNA or protein expressionis produced). The level of BBAP mRNA or protein expression in the cellscan be determined by methods described herein for detecting BBAP mRNA orprotein.

In yet another aspect of the invention, the BBAP proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with BBAP (“BBAP-binding proteins” or “BBAP-bp”) and areinvolved in BBAP activity. Such BBAP-binding proteins are also likely tobe involved in the propagation of signals by the BBAP proteins or BBAPtargets as, for example, downstream elements of a BBAP-mediatedsignaling pathway. Alternatively, such BBAP-binding proteins are likelyto be BBAP inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a BBAP protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a BBAP-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the BBAPprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified and/or produced asdescribed herein in an appropriate animal model. For example, an agentidentified and/or produced as described herein (e.g., a BBAP modulatingagent, an antisense BBAP nucleic acid molecule, a BBAP-specificantibody, or a BBAP-binding partner) can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified and/or produced asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified and/or produced by theabove-described screening assays for treatments as described herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the BBAP nucleotide sequences, describedherein, can be used to map the location of the BBAP genes on achromosome (further described in Example 1, below). The mapping of theBBAP sequences to chromosomes is an important first step in correlatingthese sequences with genes associated with disease.

Briefly, BBAP genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the BBAP nucleotidesequences. Computer analysis of the BBAP sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the BBAP sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the BBAPnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a BBAPsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the BBAP gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The BBAP sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the BBAP nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The BBAP nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The non-coding sequences of SEQ ID NO:1 cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:3 are used, a more appropriate number of primers for positiveindividual identification would be 500-2,000.

If a panel of reagents from BBAP nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial BBAP Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to non-coding regions of SEQ ID NO:1 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theBBAP nucleotide sequences or portions thereof, e.g., fragments derivedfrom the non-coding regions of SEQ ID NO:1 having a length of at least20 bases, preferably at least 30 bases.

The BBAP nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such BBAP probes can be used to identifytissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., BBAP primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining BBAP proteinand/or nucleic acid expression as well as BBAP activity, in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant BBAPexpression or activity, e.g., a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with BBAP protein, nucleic acidexpression or activity. For example, mutations in a BBAP gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby phophylactically treat an individualprior to the onset of a disorder characterized by or associated withBBAP protein, nucleic acid expression or activity e.g., a malignancysuch as a lymphoma, e.g., non-Hodgkin's lymphoma.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of BBAP inclinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of BBAPprotein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting BBAP protein ornucleic acid (e.g., mRNA or genomic DNA) that encodes BBAP protein suchthat the presence of BBAP protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting BBAP mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to BBAP mRNAor genomic DNA. The nucleic acid probe can be, for example, afull-length BBAP nucleic acid, such as the nucleic acid of SEQ ID NO:1or 3, or a portion thereof, such as an oligonucleotide of at least 15,30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to BBAP mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting BBAP protein is an antibody capable ofbinding to BBAP protein, preferably an antibody with a detectable label.Antibodies can be polyclonal or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect BBAP mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of BBAP mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of BBAP proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of BBAP genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of BBAP protein includeintroducing into a subject a labeled anti-BBAP antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting BBAP protein, mRNA, orgenomic DNA, such that the presence of BBAP protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of BBAPprotein, mRNA or genomic DNA in the control sample with the presence ofBBAP protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of BBAPin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting BBAP protein or mRNA in abiological sample; means for determining the amount of BBAP in thesample; and means for comparing the amount of BBAP in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectBBAP protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant BBAP expression or activity e.g., a malignancysuch as a lymphoma, e.g., non-Hodgkin's lymphoma. As used herein, theterm “aberrant” includes a BBAP expression or activity which deviatesfrom the wild type BBAP expression or activity. Aberrant expression oractivity includes increased or decreased expression or activity, as wellas expression or activity which does not follow the wild typedevelopmental pattern of expression or the subcellular pattern ofexpression. For example, aberrant BBAP expression or activity isintended to include the cases in which a mutation in the BBAP genecauses the BBAP gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional BBAPprotein or a protein which does not function in a wild-type fashion,e.g., a protein which does not interact with a BBAP ligand or one whichinteracts with a non-BBAP ligand.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in BBAPprotein activity or nucleic acid expression, e.g., a malignancy such asa lymphoma, e.g., non-Hodgkin's lymphoma. Alternatively, the prognosticassays can be utilized to identify a subject having or at risk fordeveloping a disorder associated with a misregulation in BBAP proteinactivity or nucleic acid expression, such as a e.g., a malignancy suchas a lymphoma, e.g., non-Hodgkin's lymphoma. Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant BBAP expression or activity in which a test sample is obtainedfrom a subject and BBAP protein or nucleic acid (e.g., mRNA or genomicDNA) is detected, wherein the presence of BBAP protein or nucleic acidis diagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant BBAP expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant BBAP expression or activity. For example, suchmethods can be used to determine whether a subject can be effectivelytreated with an agent for a e.g., a malignancy such as a lymphoma, e.g.,non-Hodgkin's lymphoma. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant BBAP expression or activity inwhich a test sample is obtained and BBAP protein or nucleic acidexpression or activity is detected (e.g., wherein the abundance of BBAPprotein or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant BBAP expression or activity).

The methods of the invention can also be used to detect geneticalterations in a BBAP gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inBBAP protein activity or nucleic acid expression, such as a e.g., amalignancy such as a lymphoma, e.g., non-Hodgkin's lymphoma. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a BBAP-protein, or the mis-expression of the BBAPgene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a BBAP gene; 2) an addition of one or morenucleotides to a BBAP gene; 3) a substitution of one or more nucleotidesof a BBAP gene, 4) a chromosomal rearrangement of a BBAP gene; 5) analteration in the level of a messenger RNA transcript of a BBAP gene, 6)aberrant modification of a BBAP gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a BBAP gene, 8) a non-wild type levelof a BBAP-protein, 9) allelic loss of a BBAP gene, and 10) inappropriatepost-translational modification of a BBAP-protein. As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a BBAP gene. A preferred biological sampleis a tissue or serum sample isolated by conventional means from asubject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the BBAP-gene (seeAbravaya et al. (1995) Nucleic Acids Res 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic DNA or mRNA) from the cells of thesample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a BBAP gene under conditions such thathybridization and amplification of the BBAP-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a BBAP gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in BBAP can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in BBAP can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the BBAP gene anddetect mutations by comparing the sequence of the sample BBAP with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be used when performingthe diagnostic assays ((1995) Biotechniques 19:448), includingsequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the BBAP gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type BBAP sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in BBAP cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a BBAP sequence,e.g., a wild-type BBAP sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in BBAP genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(orita et al. (1989) Proc Natl. Acad Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control BBAPnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method uses heteroduplex analysis to separatedouble stranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by the useof pre-packaged diagnostic kits which include at least one probe nucleicacid or antibody reagent described herein, which may be convenientlyused, e.g., in clinical settings to diagnose patients exhibitingsymptoms or family history of a disease or illness involving a BBAP genesuch as non-Hodgkin's lymphoma. Such kits can optionally includeinstructions for use.

Furthermore, any cell type or tissue in which BBAP is expressed may beused in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a BBAP protein can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent determined by a screening assay as described herein toincrease BBAP gene expression, protein levels, or upregulate BBAPactivity, can be monitored in clinical trials of subjects exhibitingdecreased BBAP gene expression, protein levels, or downregulated BBAPactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease BBAP gene expression, protein levels, ordownregulate BBAP activity, can be monitored in clinical trials ofsubjects exhibiting increased BBAP gene expression, protein levels, orupregulated BBAP activity. In such clinical trials, the expression oractivity of a BBAP gene, and preferably, other genes that have beenimplicated in, for example, a BBAP-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including BBAP, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates BBAP activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on BBAP-associated disorders (e.g., malignanciessuch as non-Hodgkin's lymphoma), for example, in a clinical trial, cellscan be isolated and RNA prepared and analyzed for the levels ofexpression of BBAP and other genes implicated in the BBAP-associateddisorder, respectively. The levels of gene expression (e.g., a geneexpression pattern) can be quantified by northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods described herein, or bymeasuring the levels of activity of BBAP or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a BBAP protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the BBAP protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the BBAP protein, mRNA, or genomic DNA inthe pre-administration sample with the BBAP protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increaseexpression or activity of BBAP to lower levels than detected, i.e., todecrease the effectiveness of the agent. According to such anembodiment, BBAP expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

D. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant BBAP expression oractivity e.g., a malignancy such as a lymphoma, e.g., non-Hodgkin'slymphoma. With regards to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the BBAP molecules ofthe present invention or BBAP modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant BBAPexpression or activity, by administering to the subject a BBAP moleculeor an agent which modulates BBAP expression or at least one BBAPactivity. Subjects at risk for a disease which is caused or contributedto by aberrant BBAP expression or activity can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the BBAP aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of BBAP aberrancy, forexample, a BBAP molecule, BBAP agonist, or BBAP antagonist can be usedto treat the subject. The appropriate agent can be determined based on,for example, the screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating BBAPexpression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a BBAP or agent that modulates one or more of theactivities of BBAP protein activity associated with the cell. An agentthat modulates BBAP protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a BBAP protein (e.g., a BBAP substrate), a BBAPantibody, a BBAP agonist or antagonist, a peptidomimetic of a BBAPagonist or antagonist, or other small molecule. In one embodiment, theagent stimulates one or more BBAP activities. Examples of suchstimulatory agents include active BBAP protein and a nucleic acidmolecule encoding BBAP that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more BBAP activities.Examples of such inhibitory agents include antisense BBAP nucleic acidmolecules, anti-BBAP antibodies, and BBAP inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a BBAP protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulate (e.g., upregulate or downregulate)BBAP expression or activity. In another embodiment, the method involvesadministering a BBAP protein or nucleic acid molecule as therapy tocompensate for reduced or aberrant BBAP expression or activity.

Stimulation of BBAP activity is desirable in situations in which BBAP isabnormally downregulated and/or in which increased BBAP activity islikely to have a beneficial effect. For example, stimulation of BBAPactivity is desirable in situations in which a BBAP molecule isdownregulated and/or in which increased BBAP activity is likely to havea beneficial effect. Likewise, inhibition of BBAP activity is desirablein situations in which BBAP is abnormally upregulated and/or in whichdecreased BBAP activity is likely to have a beneficial effect.

3. Pharmacogenomics

The BBAP molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on BBAPactivity (e.g., BBAP gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) BBAP-associated disorders (e.g.,e.g., malignancies such as non-Hodgkin's lymphoma). In conjunction withsuch treatment, pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) may be considered. Differences in metabolismof therapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a BBAP molecule or BBAPmodulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a BBAP molecule or BBAP modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) δ 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a BBAPprotein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a BBAP molecule orBBAP modulator of the present invention) can give an indication whethergene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a BBAP molecule orBBAP modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing areincorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human BBAPcDNA

In this example, the identification and characterization of the geneencoding human BBAP is described. To identify other genes whichcontribute to the observed differences in clinical outcome in DLB-CLs,yeast two-hybrid screens (Zervos et al. (1993) Cell 72:223-232) wereused to identify proteins which bind to or interact with the BALprotein. The BAL fragments that were used as baits in the yeasttwo-hybrid assay are depicted in FIG. 3. A novel BAL-associated proteintermed “B-lymphoma and BAL-associated protein” or “BBAP” has beenidentified, which specifically interacts with the BAL carboxyl terminalregion (the Moesin homologous region of BAL). The nucleotide sequenceencoding the human BBAP protein is shown in FIG. 1 and is set forth asSEQ ID NO:1. The full length protein encoded by this nucleic acidcomprises about 739 amino acids and has the amino acid sequence shown inFIG. 1 and set forth as SEQ ID NO:2. The coding region (open readingframe) of SEQ ID NO:1 is set forth as SEQ ID NO:3.

The co-association between BBAP and BAL was confirmed by co-transfectingtagged constructs (FLAG-tagged BBAP and HA-tagged BAL) into COS cells,immunoprecipitating BBAP/BAL complexes with a FLAG antibody, blottingthe immunoprecipitates, and identify BAL with an HA antibody.

Tissue Distribution of BBAP

Paired northern blot analyses of BBAL and BAL transcripts havedemonstrated that these genes are expressed at very similar levels inmultiple normal tissues and a variety of hematopoietic cell lines (seeFIG. 2).

Mapping of the BBAP Locus

To map the BBAP locus, a BBAP cDNA probe was used to screen a humangenomic DNA PAC library (RPCI1). Genomic BBAP-positive PAC clones wereused to perform FISH (fluorescence in situ hybridization) on normalhuman metaphases. In complementary experiments, a somatic cell hybridpanel was analyzed for BBAP sequences. These experiments havedemonstrated that BBAP maps to the same clone as BAL at 3q21.

Analysis of the Human BBAP Molecules

The carboxyl terminal region of the BBAP protein is 45% identical to thecarboxyl terminus of murine FXI-T1 and 47% identical to the carboxylterminus of human Deltex. FXI-T1 is the murine homologue of DrosophilaDeltex, which is differentially expressed in fractionatedX-irradiation-induced murine thymomas and contributes to FX-inducedleukemogenesis. Human Deltex (the human homologue of Drosophila Deltex)is a conserved regulator of Notch signaling and a modulator of basichelix-loop-helix (bHLH) transcription factor activity.

Amino acid analysis of the human BBAP sequence has demonstrated that thehuman BBAP comprises a C3HC4-type zinc finger motif (at amino acidresidues 561-599 of SEQ ID NO:2); three nuclear localization signals (atamino acid residues 20-26, 462-478, and 475-478 of SEQ ID NO:2); and twoshort coiled-coil domains with 2-heptad repeats (at amino acid residues347-360 and 391-404 of SEQ ID NO:2).

Human BBAP has an instability index of 50.53, which classifies thisprotein as unstable.

Taken together, these data indicate that the novel BBAP proteinco-associates with BAL in vivo, demonstrates sequence homology with acomponent of a signal pathway that functions during celldifferentiation, and contains a strong nuclear localization signal and aC3HC4-type zinc finger motif. The data further indicate that theBAL/BBAP complex may exert its effect in the nucleus. In addition, thestriking similarities in BBAP and BAL transcript abundance in thetissues examined to date indicate that BBAP and BAL may be similarlyregulated and that BBAP expression may also be risk-related in DLB-CL.

Tumorigenicity of BBAP Transfectants

BBAP's potential effects on the local growth and distant metastasis ofDLB-CL cell lines may further be determined in an in vivo murine model.Constructs containing BBAP are injected subcutaneously or via tail veininto cohorts of SCID mice. Local tumorigenicity and distant metastasiscan be scored at periodic intervals as described in Yakushijin Y. et al.(1998)Blood, 91:4282-4291.

Development of a BBAP Antibody

In order to generate a BBAP antibody, the BBAP cDNA is cloned into thepGEX expression vector (GST Gene Fusion System, Pharmacia). Aftersequencing the construct to ensure that the fusion is in frame and thatno mutations have been introduced in the BBAP sequence, bacterialcultures containing pGEX-BBAP are treated with IPTG(isopropyl-1-Thio-b-D-Galactopyearanoside) and the GST-Bal fusionprotein is induced and affinity-purified on gluthathione-5-agarosebeads. Balb-c mice are immunized with the affinity-purified GST-BBAPprotein and their spleens harvested for generation of BBAP monoclonalantibodies. Monoclonal antibodies are initially screened for reactivitywith pGEX-BBAP and not with pGEX alone using ELISA. Positive hybridomasupernatants are then screened against immunoblotted parental and BBAPDLB-CL transfectants for reactivity with the appropriate-sized BBAPprotein.

Role of BBAP in Modulating Cellular Motility and Migration

To investigate the potential role of BBAP in modulating cellularmotility and migration, BBAP is cloned into a GFP vector (PEGFP) and anuntagged expression vector (pRc-CMV) and pEGFP-BBAP, pRc-CMV BBAP, andvector only transfectants are generated in an aggressive lymphoma cellline that constitutively expresses low levels of BBAP. The effects ofBBAP overexpression on the migration of these transfectants isinvestigated using a transwell system. In initial experiments, BBAP-GFPor GFP-only transfectants are plated in the upper chamber and analyzedfor migration to the lower chamber in the presence of the hematopoieticchemoattractant factor, stromal derived factor 1-α (SDF-1α).

Example 2 Expression of Recombinant BBAP Protein in Bacterial Cells

In this example, human BBAP is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, BBAP isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-BBAP fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 3 Expression of Recombinant BBAP Protein in COS Cells

To express the BBAP gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire BBAP protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the BBAP DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the BBAP codingsequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the BBAP coding sequence. The PCR amplified fragmentand the pcDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the BBAP gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the BBAP-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, N.Y., 1989.The expression of the BBAP polypeptide is detected by radiolabelling(³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine).The culture media are then collected and the cells are lysed usingdetergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50mM Tris, pH 7.5). Both the cell lysate and the culture media areprecipitated with an HA specific monoclonal antibody. Precipitatedpolypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the BBAP coding sequence is cloneddirectly into the polylinker of the pcDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the BBAPpolypeptide is detected by radiolabelling and immunoprecipitation usinga BBAP specific monoclonal antibody.

Example 4 Subcellular Localization of BBAP and BAL Protein

To gain additional insight into the function of BBAP, a FLAG-tagged BBAPand BAL fused to green fluorescence protein (GFP) were used to determinethe subcellular localization of these proteins. COS7 cells weretransfected with the FLAG-tagged BBAP and, subsequently,immunofluorescent staining was performed using an anti-Flag and aRhodamine Red-X-conjugated secondary antibody. Based on the foregoinganalysis, it was determined that, contrary to the BAL protein, BBAPlocalizes primarily to the cytoplasm. Use of an anti-BBAP monoclonalantibody confirmed the cytoplasmic staining, indicating thatlocalization of BBAP to the cytoplasm is not an artifact of fusion toFLAG or overexpression. As indicated in previous studies, BAL localizedto the nucleus of COS7 cells transfected with a BAL-GFP construct in theabsent of BBAP protein. However, when COS7 cells were co-transfectedwith BAL-GFP and Flag-tagged BBAP, BAL co-localized with BBAP in thecytoplasm. The foregoing data indicate that BBAP plays an important rolein determining the subcellular localization of BAL.

Subcellular fractionation of the DLB-CL cell lines shows that both BBAPand BAL are primarily localized in the cytoplasm, but also exist in thenucleus. The concentration of the two proteins is very similar in bothsubcellular organelles (nucleus and cytoplasm) in a variety of DLB-CLcell lines (e.g., DHL4, DHL6, DHL7, DHL10), indicating that theendogenous subcellular localization of the BBAP and BAL proteins issimilar, unlike the localization of BAL or BBAP when each is presentalone in a cell.

Example 5 Co-Association of BBAP and Human DTX1

Studies of human DTX1 (human Deltex) and murine MDTX family members haveshown that DTX proteins form homotypic and heterotypic multimers. Asindicated above, the BBAP and DTX1 C-termini are highly homologous. Todetermine whether there is a physical association between BBAP and DTX1,COS7 cells were co-transfected with Flag-tagged BBAP and Myc-taggedDTX1, BBAP was immunoprecipitated with an anti-flag antibody, and theprecipitates were analyzed by western blotting. DTX1-Myc was clearlydetected in BBAP-Flag immunoprecipitates of cells that wereco-transfected with both constructs. The foregoing data confirm thatBBAP and DTX1 co-associate in vivo.

Example 6 Suppression of the E47 Promoter

Activation of Notch signaling in cultured mammalian cells results in thesuppression of transcriptional activation by a human bHLH protein, E47(Ordentlich, P. et al. (1998) Mol. Cell. Biol. 18, 22302239). Forexample, the transcription of an E47-responsive reporter gene isspecifically suppressed when E47 is co-expressed with an activated formof human Notch1 or DTX1 (Ordentlich, P. et al. supra and Matsuno, K. etal. (1998) Nat. Genet. 19, 74-78). In this experiment, the effect of theBBAP protein on E47 activity was evaluated using a Luciferase reporterconstruct driven by an E47-responsive promoter. As shown in FIG. 4, therelative activity of the E47-responsive reporter was suppressedsignificantly when E47 was co-expressed with human DTX1, in a dosagesensitive fashion. Under the same conditions, E47 activity was alsoinhibited when E47 was co-expressed with human BBAP. However, theinhibition of E47 activity by BBAP was weaker than the activityinhibition observed with DTX1, and the suppression of reporter activitywas less dosage dependent. The foregoing observations indicate that theBBAP protein has an inhibitory effect on E47 activity that is similar tothat seen with human DTX1. Unlike human DTX1, BBAP's effect on E47 maybe indirect.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide which is encoded by a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
 2. Theisolated polypeptide of claim 1, comprising the amino acid sequence ofSEQ ID NO:2.
 3. The polypeptide of claim 1, further comprisingheterologous amino acid sequences.
 4. A method of producing apolypeptide comprising culturing a host cell transfected with anexpression vector comprising a nucleic acid molecule encoding apolypeptide of claim 1 in an appropriate culture medium to, thereby,produce the polypeptide.
 5. An isolated polypeptide comprising the aminoacid sequence of SEQ ID NO:2.